12868
J. Am. Chem. Soc. 1996, 118, 12868-12869
Direct Detection by 15N NMR of the Tryptophan Tryptophylquinone Aminoquinol Reaction Intermediate of Methylamine Dehydrogenase G. Reid Bishop,1a Edward J. Valente,1b Tracy L. Whitehead,1c Kenneth L. Brown,1c Rickey P. Hicks,*,1c and Victor L. Davidson*,1a Department of Biochemistry The UniVersity of Mississippi Medical Center Jackson, Mississippi 39216-4505 Department of Chemistry, Mississippi College Clinton, Mississippi 39058 Department of Chemistry, Mississippi State UniVersity Mississippi State, Mississippi 39762 ReceiVed June 27, 1996 Redox-active cofactors, derived from post-translational modifications of amino acid side chains, are emerging as critical participants in many enzyme-catalyzed reactions.2 Amino-acidderived quinone-containing prosthetic groups,2d,3 such as tryptophan tryptophylquinone2c (TTQ) and 2,4,5-trihydroxyphenylalanine2b quinone (TPQ), have been suggested, although not directly shown, to be chemically modified by a substrate during their catalytic cycles. We present 15N NMR data which directly confirms the covalent modification, by substrate nitrogen (N) of TTQ in methylamine dehydrogenase (MADH), and demonstrate that 15N NMR may be used to monitor the fate of substrate 15N throughout the catalytic cycle. MADH3 catalyzes the oxidative deamination of primary amines to the corresponding aldehyde, ammonium, and two electrons that are conveyed in ViVo by sequential one-electron transfers to a type-1 copper protein, amicyanin.4 These reactions of MADH are mediated by TTQ which is derived by posttranslational modification of two tryptophan residues (Figure 1A).2c,5 Chemical reaction occurs at the C6 carbonyl.6 It has been postulated that reduction of TTQ by substrate results in a stable aminoquinol intermediate7 (Figure 1A) by a transaminase type mechanism.3c,7a Alternatively, O-quinol TTQ with hydroxyl groups at C6 and C7 is generated by titration of MADH with dithionite.8 Dithionite- and substrate-reduced MADH exhibit different reactivities with amicyanin7b,c supporting the idea that substrate N is covalently incorporated9 into TTQ and * Corresponding authors: V. L. Davidson, Department of Biochemistry, The University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505, telephone 601-984-1515, e-mail davidson@ fiona.umsmed.edu; R. P. Hicks, Department of Chemistry, Mississippi State University, P.O. Box 9573, Mississippi State, MS 39762, telephone 601325-3584, e-mail
[email protected]. (1) (a) The University of Mississippi Medical Center. (b) Mississippi College. (c) Mississippi State University. (2) (a) Klinman, J. P., Ed. Methods Enzymol. 1995, 258. (b) Janes, S. M.; Mu, D.; Wemmer, D.; Smith, A. J.; Kaur, S.; Maltby, B.; Burlingame, A. L.; Klinman, J. P. Science 1990, 248, 981. (c) McIntire, W. S.; Wemmer, D. E.; Christoserdov, A.; Lidstrom, M. E. Science 1991, 252, 817. (d) Klinman, J. P.; Mu, D. Annu. ReV. Biochem. 1994, 63, 299. (3) (a) Davidson, V. L., Ed.; Principles and Applications of Quinoproteins; Marcel Dekker, Inc.: New York, 1993; p 73. (b) Davidson, V. L. Methods Enzymol. 1990, 188, 241. (c) Davidson, V. L.; Brooks, H. B.; Graichen, M. E.; Jones, L. H.; Hyun, Y. L. Methods Enzymol. 1995, 258, 176. (4) Husain, M.; Davidson, V. L. J. Biol. Chem. 1985, 260, 14626. (5) Christoserdov, A. Y.; Tsygankov, Y. D.; Lidstrom, M. E. Biochem. Biophys. Res. Commun. 1990, 172, 211. (6) Huizinga, E. G.; van Zanteen, B. A. M.; Duine, J. A.; Jongejan, J. A.; Hullema, F.; Wilson, K. S.; Hol, W. G. J. Biochemistry 1992, 31, 9789. (7) (a) Brooks, H. B.; Jones, L. H.; Davidson, V. L. Biochemistry 1993, 32, 2725. (b) Bishop, G. R.; Davidson, V. L. Biochemistry 1995, 34, 12082. (c) Bishop, G. R.; Brooks, H. B.; Davidson, V. L. Biochemistry 1996, 35, 8948. (8) Husain, M.; Davidson, V. L.; Gray, K. A.; Knaff, D. B. Biochemistry 1987, 26, 4139. (9) Warncke, K.; Brooks, H. B.; Babcock, G. T.; Davidson, V. L.; McCracken, J. J. Am. Chem. Soc. 1993, 115, 6464.
S0002-7863(96)02185-3 CCC: $12.00
Figure 1. Reductive and oxidative half-reactions of MADH. A. Reduction is hypothesized to result in covalent modification of the C6 carbon of TTQ by substrate-derived N to form the aminoquinol. The protonation state of the aminoquinol is unknown. B. The two-electron oxidation of aminoquinol TTQ is accompanied by hydrolysis of the substrate N and results in a replacement of N by solvent-derived O.
released only after reoxidation. This mechanism has been disputed, and differences in these reactivities have alternatively been ascribed to the presence of noncovalently bound product ammonia interacting with the C6 oxygen of the O-quinol.10 Here, we use CH315NH2 and 15N NMR spectroscopy to observe directly the fate of the substrate-derived 15N. Purification of MADH from Paracoccus denitrificans was as previously described.3b CH3NH3Cl and NH4Cl (15N enriched to 99%) were purchased from CDN Isotopes and benzamide (15N enriched to 99%) from Cambridge Isotope Laboratories. Experiments were performed in 10 mM potassium phosphate buffer, pH 7.5, plus 200 mM KCl, with 10% v/v D2O for NMR signal lock at 25 °C. MADH (3 mM) was reacted with 20fold excess CH315NH3Cl. No significant loss of MADH activity was observed in conditions mimicking those of the NMR experiments (up to 3 d at 25 °C). Samples were analyzed in 10 mm NMR tubes on a General Electric Omega-400 at 40.55 MHz with proton decoupling at 400.13 MHz. Spectral data were acquired with a width of 20 000 Hz and 128-30000 scans per spectrum and processed using 1.0 Hz line broadening. Chemical shifts (δ) of 15N were referenced to 110.0 ppm for external 15N-labeled benzamide but reported against liquid NH3 (δ ) 0.0 at 40.55 MHz).11 15N spectra were collected over a 480 ppm range to assure observation of all N species. 15N NMR relaxation times are long for solutes and short for protein-bound N. Figure 2A shows that CH315NH3Cl yields a signal at δ ) 35 ppm with a long pulse delay (120 s) which decreases on a short pulse delay (1 s) due to saturation of the slowly relaxing species (data not shown). After reduction of MADH by excess CH315NH3Cl, two peaks are observed with a 1 s pulse delay (Figure 2B).12 The upfield signal at δ ) 35 ppm exhibits a negative NOE and is characteristic of CH315NH3+ (Table 1). The new signal observable optimally at a short pulse delay (1 s) appears downfield at δ ) 54 ppm. Anilino 15N chemical shifts are downfield by about 25 ppm from alkylamines under similar conditions13 (Table 1). The signal at δ ) 54 ppm is consistent with 15N covalently attached to an (10) (a) Gorren, A. C. F.; de Vries, S.; Duine, J. A. Biochemistry 1995, 34, 9748. (b) Gorren, A. C. F.; Moenne-Loccoz, P.; Backes, G.; de Vries, S.; Sanders-Loehr, J.; Duine, J. A. Biochemistry 1995, 34, 12926. (11) Srinivasen, P. R.; Lichter, R. L. J. Magn. Reson. 1977, 28, 277.
© 1996 American Chemical Society
Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12869 Table 1. 15 NMR Experimental and Reference Chemical Shifts for Model Compounds compd NH4Cl
concentrations and solvent
saturated in 90% H2O/10% D2O 1 M in 10% D2O/90% bufferb CH3NH3Cl saturated in 50% D2O/50% H2O 1 M in 10% D2O/90% bufferb PhNH3Clc concentrated in 90% H2O/10% D2O 40% in 10% D2O/90% H2O PhCONH2d saturated in acetone, +10% D2O
δ15N (ppm)a
ref
30 35 33 35 55 60 110
15 this work 16 this work 13b this work this work
a Nearest ppm referenced to NH3(l) where δ ) 0.0 (refs 11 and 15). Buffer is 10 mM potassium phosphate in H2O, pH 7.5 with 200 mM KCl. c Natural abundance 15N; all others enriched. d External reference in this work.
b
Figure 2. 15N NMR spectra depicting the fate of substrate-derived N during the catalytic cycle of MADH. A. CH315NH3Cl in the absence of enzyme (acquisitions per free induction decay [FID] ) 128, pulse delay ) 120 s, acquisition time ) 2.2 s). B. MADH reduced by excess CH315NH3Cl, (acquisitions per FID ) 30 000, pulse delay ) 1 s, acquisition time ) 2.2 s). C. Substrate-reduced MADH after extensive dialysis (acquisitions per FID ) 30 000, pulse delay ) 1 s, acquisition time ) 2.2 s). D. MADH after reoxidation by excess PES (acquisitions per FID ) 5000, pulse delay ) 1 s, acquisition time ) 2.2 s). E. Sample from D with added 15NH4Cl (acquisitions per FID ) 5000, pulse delay ) 60 s, acquisition time ) 1.6 s).
aromatic ring (i.e., aminoquinol, Figure 1). With a short delay (1.0 s), δ ) 54 ppm accumulates, implying the signal relaxes quickly as expected for 15N covalently bound to MADH. Additional verification that δ ) 54 ppm is due to nonexchangeable N was obtained from dialysis of the sample (Figure 2C). The negative peak at δ ) 35 ppm, attributed to free unreacted CH315NH3+, is absent, whereas the δ ) 54 ppm signal is (12) Due to their high viscosity, protein samples were somewhat difficult to shim, and observed line widths were variable. Thus, an analytic interpretation of observed line widths is not appropriate, particularly when sample spectra obtained from different runs are compared (e.g., Figures 2B,C). On average, line widths of δ ) 54 were 3-fold greater than those of δ ) 35 from free CH315NH3+ and 15NH4+ (Figures 2A,D,E). The negative signal at δ ) 35 in Figure 2B is noticeably broadened. This suggests that the free CH315NH3+ may be in slow exchange with protein bound CH315NH3+.
retained. To verify that CH315NH3+ was absent and not unobserved due to saturation of a slowly relaxing species, the pulse delay was increased to 60 s. No signal at δ ) 35 ppm was detected even after 10 h of spectral acquisition. Thus, the signal at δ ) 54 ppm describes 15N derived from a substrate which is covalently bonded to an aromatic system and attached to a protein framework. Immediately after treatment of the reduced MADH sample with a 100-fold excess of the oxidant phenazine ethosulfate3b (PES), one observes (Figure 2D) the disappearance of δ ) 54 ppm and the emergence of δ ) 35 ppm. The δ ) 35 ppm signal must be due to a slowly relaxing species (i.e., free rather than protein-bound N), since spectral accumulation occurs optimally at a long (60 s) pulse delay. This is consistent with free product 15NH4+ (Table 1). To confirm this, 15NH4Cl was added to the sample which led to an increase in δ ) 35 ppm (Figure 2E). Dialysis of the protein results in loss of all 15N signals. The dialysate contains 15NH4+, judged by a signal at 35 ppm resolved positively by a long pulse delay (not shown). Studies with model TPQ compounds suggested that reduced triol preparations may form (alkylamino)resorcinols via a redox cycling mechanism involving condensation of amines with trace amounts of hydroxyquinone in the triol preparations14 or with cyclohexadienone tautomers. To confirm that the δ ) 54 ppm species is not due to covalent modification of O-quinol TTQ by excess CH315NH3+, the experiments described in Figure 2 were repeated with dithionite-reduced MADH under identical conditions. No signal at δ ) 54 ppm was observed, only the slow relaxing signal at δ ) 35 ppm corresponding to free methylamine. These data are consistent with and confirm the mechanisms presented in Figure 1, where quinone TTQ is covalently modified by substrate N to form a stable and relevant aminoquinol intermediate. Reoxidation of the aminoquinol to the quinone, results in loss of covalent N as free product 15NH4+. These data also demonstrate the utility of 15N NMR spectroscopy as a tool for monitoring the reaction steps of enzymes which catalyze transformations of biological amines. Acknowledgment. We thank Dr. J. L. Eglin, R. Williams, and A. B. Dribben. Supported by NIH grant GM-41574, NSF grant CHE92124521, and Mississippi State University. JA9621859 (13) (a) Axenrod, T.; Pregosin, P. S.; Wieder, M. J.; Becker, E. D.; Bradley, R. B.; Milne, G. W. A. J. Am. Chem. Soc. 1971, 93, 6536. (b) Psota, L.; Franzen-Seveking, M.; Turnier, J.; Lichter, R. L. Org. Magn. Reson. 1978, 11, 401. (14) Wang, F.; Bae, J.-Y.; Jacobson, A. R.; Lee, Y.; Sayre, L. M. J. Org. Chem. 1994, 59, 2409. (15) Stefaniak, L.; Webb, G. A.; Witanowski, M. Annu. Rep. NMR Spectrosc. 1981, 11B, 144. (16) Preece, N. E.; Nicholson, J. K.; Timbrell, J. A. Biochem. Pharmacol. 1991, 41, 1319.
